To begin, obtain paraffin-embedded slice of mouse lung tissue and place it in a 65 degree Celsius oven for 60 minutes. For staining, immerse the slide with the tissue section sequentially in the staining jars containing appropriate solutions. Dilute the modified sodium citrate antigen retrieval solution 50-fold with deionized water.
After preheating the antigen extract in the microwave on high power, place the slide in it for 15 minutes to boil. After the slide cools to room temperature, wash it two times with PBS for five minutes each. Then permeabilize the tissue with 0.25%triton two times for 10 minutes each.
Encircle the tissue with an immunohistochemistry pen and incubate the slide with 5%BSA at room temperature for one hour. Next, dilute the isolectin B4, or IB4, with 1%BSA to a ratio of 1 to 50. After removing excess water, incubate the tissue section with 50 to 100 microliters of the diluted IB4 overnight at four degrees Celsius.
Wash the slides three times with PBS for five minutes each and permeabilize with 0.25%triton for 10 minutes. Then incubate the tissue section with 50 to 100 microliters DAPI at room temperature for five minutes. After three PBS washes, apply a drop of anti-fade mounting medium to the slide, avoiding light exposure.
Mount the air-dried slide under a fluorescence microscope to capture images of the nucleus and target signals. This protocol enables precise localization and observation of pulmonary microvasculature using IB4 for staining micro endothelial cells in different mouse lung lobes under a fluorescence microscope.
